Recent studies by us and other investigators have shown a role for 1,25- dyhydroxyvitamin D3 (1,25(OH)D) and calcium in epidermal differentiation. Our studies support a relationship between differentiation and endogenous generation of vitamin D metabolites by epidermal cells (keratinocytes). In this proposal, we plan to evaluate the regulation of endogenous vitamin D metabolite generation and determine the effect of endogenously produced vitamin D metabolites on keratinocyte differentiation focussing on their interaction with calcium. The role of different vitamin D metabolites on differentiation will be determined by evaluating the ability of specific inhibitors of the 1 and 24-hydroxylases to block the induction of transglutaminase and involucrin by 250HD and its products 1,25(OH)2D and 24,25 (OH)2D. We will assess the interaction of 1,25(OH)2D and calcium on keratinocyte differentiation by determining the ability of 1,25(OH)2D to modulate the regulation by calcium of ivolucrin and transglutaminase gene transcription, message stability and protein production. We will then explore the mechanisms by which 1,25(OH)2D increases intracellular calcium, examining the role of calmodulin (CaM), CaM binding proteins, membrane lipids and membrane fluidity. Finally, we will determine whether keratinocytes are capable of producing 250HD and 1,25(OH)2D from endogenously produced vitamin D using UV irradiation, and assess the regulation of this pathway by cytokines also produced by keratinocytes in response to UV exposure.